PROJECT SUMMARY Periodontitis, a microbial-driven, destructive disease of the tissues surrounding the teeth, occurs in approximately 50% of the population and is associated with several debilitating systemic conditions (including vascular and lung diseases, diabetes mellitus, and pre-term birth). Patients who smoke have increased susceptibility to periodontitis and are more likely than non-smokers to display severe disease and to be refractory to treatment. Indeed, the most recent epidemiological evidence suggests that tobacco smoking accounts for the majority of destructive periodontal disease cases in developed nations. Smoking enhances infection rates and enriches numbers of the keystone periodontal pathogen, Porphyromonas gingivalis. However, the mechanisms underlying this phenomenon are in need of elucidation. We plan to generate whole genome transposon sequencing (TnSeq) libraries for two P. gingivalis strains (ATCC 33277 and W83) to be employed in order to (i) identify genes that are putatively essential for P. gingivalis to survive cigarette smoke exposure in vitro; (ii) to validate that TnSeq-identified genes are indeed essential by generating single gene deletion mutants and monitoring their ability to grow planktonically and in biofilms under cigarette-induced stress; (iii) to determine the in vivo relevance of such mutations in a murine model of smoke-exacerbated periodontitis; (iv) to establish the distribution of essential genes in multiple low-passage clinical P. gingivalis isolates; (v) to characterize the function of those genes confirmed to be requisite for survival of tobacco-induced stress both in vitro and in vivo and (vi) to identify, in silico, essential gene orthologues in other bacteria whose virulence is also enhanced in tobacco smokers. There are several potential translational benefits to a successful R01, which include a better understanding of how P. gingivalis thrives in a tobacco-toxin rich environment; future therapeutic targeting of essential genes to control P. gingivalis infection in smokers; potential identification of novel vaccine targets for prevention or control of P. gingivalis infection; alternate treatment regimens for smokers based on mechanistic insight into smoke-induced and/or exacerbated periodontal diseases; and the establishment of an essential gene database for tobacco-enhanced pathogens that will facilitate the identification of common bacterial strategies for surviving tobacco smoke exposure, thus, broadening the significance of the research beyond the oral cavity.